Bottom Line:
Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis.Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals.However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation.

ABSTRACTGenetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites--recombination hotspots--through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F(1) hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)--the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.

Mentions:
Despite the clear critical role of PRDM9 in the initiation of genetic recombination meiotic DSBs are present in Prdm9 knockout mice9. Remarkably, we found that initiation of recombination in these mice is not random or uniform, but is still clustered in hotspots. The vast majority (99%) of these hotspots do not overlap the hotspots detected in any of the wild type strains, nevertheless, 94% of Prdm9−/− hotspots still overlap H3K4me3 marks. Most of these marks (92%) are present in wild type testis and almost half are not specific to germline cells (Supplementary Fig. 5d). As H3K4me3 marks outnumber DSB hotspots, they do not appear to be sufficient for hotspot formation (Supplementary Fig. 6). H3K4me3 is a general mark of active transcription primarily associated with gene promoters16, enhancers17,18 and possibly other functional genomic elements. We found that in the absence of PRDM9 and PRDM9-introduced H3K4me3 marks, recombination hotspots are re-routed to these alternative H3K4me3 sites (Fig. 2a). Almost half (44%) of recombination hotspots in the Prdm9−/− mice localize to the promoters of annotated genes compared to just 3% in wild type. Unlike in wild type mice, promoter-associated hotspots in Prdm9−/− are relatively strong (Supplementary Fig. 7) and therefore, account for the majority of DSB-derived sequencing reads (62%; Fig. 2b). The H3K4me3 signal at promoters that overlap hotspots in Prdm9−/− is almost 100-fold stronger than at those that do not (median: 350 tags and 5 tags, respectively, P < 10−200, Wilcoxon test). Furthermore, promoter-overlapping hotspots are slightly enriched at genes expressed in early meiotic prophase (P = 10−138, one-sided binomial test; Supplementary Fig. 8). Prdm9−/− hotspots that are not associated with annotated promoters still likely represent DSBs formed at important genomic elements as they exhibit higher than expected sequence conservation (Supplementary Fig. 9). Indeed, 40% of these hotspots overlap histone crotonylation sites, a recently discovered marker of promoters and enhancers19 (see Supplementary Methods). The distribution of hotspots around transcription start sites (TSSs) in Prdm9−/− mice is reminiscent of that in S. cerevisiae where most hotspots are found at promoters20,21. However, while DSBs at yeast promoters form in the upstream, nucleosome-depleted region20,21, hotspots in Prdm9−/− mice form preferentially at the most frequently H3K4 trimethylated nucleosome at the +1 position, just downstream of the TSS (Fig. 2c).

Mentions:
Despite the clear critical role of PRDM9 in the initiation of genetic recombination meiotic DSBs are present in Prdm9 knockout mice9. Remarkably, we found that initiation of recombination in these mice is not random or uniform, but is still clustered in hotspots. The vast majority (99%) of these hotspots do not overlap the hotspots detected in any of the wild type strains, nevertheless, 94% of Prdm9−/− hotspots still overlap H3K4me3 marks. Most of these marks (92%) are present in wild type testis and almost half are not specific to germline cells (Supplementary Fig. 5d). As H3K4me3 marks outnumber DSB hotspots, they do not appear to be sufficient for hotspot formation (Supplementary Fig. 6). H3K4me3 is a general mark of active transcription primarily associated with gene promoters16, enhancers17,18 and possibly other functional genomic elements. We found that in the absence of PRDM9 and PRDM9-introduced H3K4me3 marks, recombination hotspots are re-routed to these alternative H3K4me3 sites (Fig. 2a). Almost half (44%) of recombination hotspots in the Prdm9−/− mice localize to the promoters of annotated genes compared to just 3% in wild type. Unlike in wild type mice, promoter-associated hotspots in Prdm9−/− are relatively strong (Supplementary Fig. 7) and therefore, account for the majority of DSB-derived sequencing reads (62%; Fig. 2b). The H3K4me3 signal at promoters that overlap hotspots in Prdm9−/− is almost 100-fold stronger than at those that do not (median: 350 tags and 5 tags, respectively, P < 10−200, Wilcoxon test). Furthermore, promoter-overlapping hotspots are slightly enriched at genes expressed in early meiotic prophase (P = 10−138, one-sided binomial test; Supplementary Fig. 8). Prdm9−/− hotspots that are not associated with annotated promoters still likely represent DSBs formed at important genomic elements as they exhibit higher than expected sequence conservation (Supplementary Fig. 9). Indeed, 40% of these hotspots overlap histone crotonylation sites, a recently discovered marker of promoters and enhancers19 (see Supplementary Methods). The distribution of hotspots around transcription start sites (TSSs) in Prdm9−/− mice is reminiscent of that in S. cerevisiae where most hotspots are found at promoters20,21. However, while DSBs at yeast promoters form in the upstream, nucleosome-depleted region20,21, hotspots in Prdm9−/− mice form preferentially at the most frequently H3K4 trimethylated nucleosome at the +1 position, just downstream of the TSS (Fig. 2c).

Bottom Line:
Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis.Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals.However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation.

ABSTRACTGenetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites--recombination hotspots--through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F(1) hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)--the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.